Fig 1: TMPRSS2 has no effects on viral infectivity of pseudoviruses that infect HEK-293T-ACE2-TMPRSS2. A. Pseudoviruses bearing the indicated SARS-CoV-2 spike P681 mutations were used to transduce HEK-ACE2 cells and HEK cells that express HA-TMPRSS2. The human protease was transfected into cells 48hr earlier and its expression was verified in infected cells by FACS (panel A). Equal viral loads were normalized based on p24 protein levels. 48hr post transduction, cells were harvested and their luciferase readouts were monitored. Bar graphs show mean values ± SD error bars of three independent experiments. Measured statistical significance was calculated between experiments by a two-tailed Student’s t test ***P=0.001. B. Control HEK-293 non-transfected cells and cells that stably express TMPRSS2-HA were dissociated by EDTA treatment, and washed with PBS-0.01% Tritin washing buffer. Cells were then incubated with 0.04% triton for 10 min on ice, washed and blocked with 4% FCS in PBS. Cells were then incubated with anti-HA IgG (5 µg/ml, Abcam ab9110) for 45 min on ice. After x3 washes with washing buffer, the cells were incubated with Rhodamine-Red labeled goat anti-Rabbit IgG antibody (1:200; Jackson 111-296-003), washed x3 times with washing buffer and analyzed by FACS.